[Studies on the pathogenesis of type 1 diabetes mellitus--immunochemical studies with monoclonal islet cell surface antibody using hybridization of spleen lymphocytes from non-obese diabetic mice].

The production of monoclonal antibodies to islet cell surface antigens, using hybridization of spleen lymphocytes from non-obese diabetic (NOD) mice has been reported previously from our laboratory. In the present study, the immunochemical characteristics of the monoclonal antibody (3A4) have been investigated using In-111 cells, a virus-induced insulinoma cell line derived from the Syrian golden hamster as target cells. The antibody 3A4 could be visually detected in the immunoenzymatic labelling of the surface of In-111 cells. To identify the molecular weight of target specific antigens reacting with 3A4, 125I-surface labelled In-111 cells were solubilized and extracts were absorbed with 3A4. The immunoprecipitates were subjected to polyacrylamide gel electrophoresis and autoradiography. 3A4 recognized two major polypeptides with apparent molecular weights of radioactive 64K and inactive 28K daltons. In order to evaluate antibody-mediated cytotoxic mechanisms of 3A4, complement-dependent antibody-mediated cytotoxicity (C'AMC) and antibody-dependent cellular cytotoxicity (ADCC) were tested, using a method of specific chromium release. In the study for C'AMC, even though over wide ranges of antibody concentration and rabbit complement, purified 3A4 had no apparent cytotoxic effects on In-111 cells. On the other hand, significant ADCC was observed at 10 micrograms/ml antibody concentration and 1:40 target: effector cell ratio. Finally, the effect of 3A4 on glucose-stimulated insulin release in isolated rat islets was examined. At 16.7 mM glucose concentration, 3A4 significantly inhibited the insulin release in the absence or presence of complement. Therefore, 3A4 can not only bind but also be active to the target cells in the cytotoxicity and suppression of insulin release, and it can be a useful tool to clarify the pathogenesis of type 1 diabetes mellitus. Furthermore, these results suggest that the relationship between islet cell surface antibody and cell-mediated immunity, especially immunoresponse against certain antigenic determinants on pancreatic B cells, seems to be important in the pathogenesis of type 1 diabetes mellitus.